MicroRNAs in the hypothalamus.

MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting in specific cleavage and subsequent degradation of the mRNA or by translational repression resulting in specific inhibition of protein synthesis. An increasing amount of evidence shows that miRNAs control a large number of biological processes and there exists a direct link between miRNAs and disease. miRNA molecules are abundantly expressed in tissue-specific and regional patterns and have been suggested as potential biomarkers, disease modulators and drug targets. The central nervous system is a prominent site of miRNA expression. Within the brain, several miRNAs are expressed and/or enriched in the region of the hypothalamus and miRNAs have recently been shown to be important regulators of hypothalamic control functions. The aim of this review is to summarize some of the current knowledge regarding the expression and role of miRNAs in the hypothalamus.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

Neurotransmitters in key neurons of the hypothalamus that regulate feeding behavior and body weight.

During the last two decades attention has been focussed on the role of different neuropeptides in hypothalamic control of feeding behavior. Several hypothalamic peptides that participate in the control of ingestive behavior are produced in neuronal cell bodies of the arcuate nucleus and/or the lateral hypothalamic area. Apart from producing orexigenic or anorexigenic compounds of peptidergic nature, these neurons also produce excitatory and inhibitory amino acid neurotransmitters. The role of GABA and glutamate in regulating energy balance has received less attention in comparison to neuropeptides. The arcuate nucleus-median eminence area, a region with a weak blood-brain barrier, contains at least two neuronal cell populations that exert opposing actions on energy balance. The majority of the neurons located in the ventromedial aspect of the arcuate nucleus, which produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP), contain in addition the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter (VGAT), thereby supporting their GABAergic nature. Some neurons producing pro-opiomelanocortin (POMC)- and cocaine- and amphetamine-regulated transcript (CART), located in the ventrolateral division of the arcuate nucleus have recently been reported to contain the vesicular glutamate transporter 2 (VGLUT2), a marker for glutamatergic neurons, and the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) as well as the vesicular ACh transporter (VAChT), supporting also a cholinergic phenotype. In the lateral hypothalamic area, hypocretin/orexin neurons express VGLUT1 or VGLUT2, but not GAD, whereas some melanin-concentrating hormone (MCH) cells contain GAD. These observations support the view that several classical transmitters, relatively neglected feeding transmitters candidates, are present in key neurons that regulate body weight and consequently may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in regulation of energy balance.

Hypothalamic proopiomelanocortin (POMC) neurons have a cholinergic phenotype.

Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Neuropeptide Y (NPY)-producing neurons in the arcuate nucleus stimulate food intake, whereas arcuate nucleus neurons that release the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) potently reduce food intake. Relatively little attention has been focused on classical neurotransmitters in regulation of food intake. Here, we have investigated the potential presence of acetylcholine (ACh) in NPY- and POMC-containing neuronal populations of the arcuate nucleus. Antisera to proteins required for cholinergic neurotransmission, including choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), were employed in double-labeling immunohistochemical experiments. In colchicine-treated rats, ChAT- and VAChT-immunopositive cell bodies were located in the ventral aspect of the arcuate nucleus. ChAT and VAChT immunoreactivities were demonstrated in alpha-MSH- and cocaine- and amphetamine-regulated transcript (CART)-containing cell bodies of the arcuate nucleus, whereas cell bodies containing NPY or agouti-related peptide (AGRP) were distinct from VAChT-immunoreactive neuronal perikarya. VAChT immunoreactivity was also present in a large number of alpha-MSH-containing nerve fiber varicosities throughout the central nervous system. In the commissural part of the nucleus tractus solitarius, no alpha-MSH-containing cell bodies were found to have ChAT or VAChT immunoreactivity. The presence of markers for cholinergic neurotransmission in a subpopulation of hypothalamic POMC/CART neurons suggests co-release of ACh with peptides derived from the POMC precursor and CART. The results indicate a role for ACh in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin.

Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight.

After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-associated protein termed excitatory amino acid transporter 3 (EAAT3). Following entry into the nerve terminal, glutamate is pumped into synaptic vesicles by a vesicular transport system. Three different vesicular glutamate transporter proteins (VGLUT1-3) representing unique markers for glutamatergic neurons were recently characterized. The presence of EAAT3, glutaminase and VGLUT1-3 was examined in mouse, rat and rabbit species at mRNA and protein levels in hypothalamic neurons which are involved in the regulation of body weight using in situ hybridization and immunohistochemistry. EAAT3 and glutaminase mRNAs were demonstrated in all parts of the arcuate nucleus in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamic area. VGLUT1 mRNA was present in the magnocellular lateral hypothalamic nucleus. VGLUT2 mRNA was demonstrated in a subpopulation of neurons in the arcuate nucleus and in the ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamic area. Few VGLUT3 mRNA expressing neurons were scattered throughout the medial and lateral hypothalamus. EAAT3-like immunoreactivity (-li) was demonstrated in glutamate, neuropeptide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanin-concentrating hormone and orexin-immunoreactive (-ir) neurons. VGLUT2-li could only be demonstrated in POMC- and CART-ir neurons of the ventrolateral arcuate nucleus. The results show that key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NPY/AGRP neurons situated in the ventromedial part of the arcuate nucleus are mainly GABAergic, it is shown that several anorexigenic POMC/CART neurons of the ventromedial arcuate nucleus are most likely glutamatergic [corrected].

5-HT1A receptor immunoreactivity in hypothalamic neurons involved in body weight control.

Serotonin (5-hydroxytryptamine; 5-HT) is a regulator of feeding behavior. The effect of serotonin on food intake is believed to be primarily mediated via 5-HT(1A) and 5-HT(2C) receptors, which both are expressed in hypothalamic regions implicated in regulation of feeding behavior. Using an antiserum to the 5-HT(1A) receptor, immunoreactive neurons were observed in the rat supraoptic, paraventricular, arcuate and ventromedial nuclei and lateral hypothalamic area. 5-HT(1A) receptor immunoreactivity was demonstrated in neuropeptide Y-, agouti-related peptide-, proopiomelanocortin- and cocaine- and amphetamine-regulated transcript-containing neurons of the arcuate nucleus. In the lateral hypothalamus, 5-HT(1A) receptor immunoreactivity was observed in melanin-concentrating hormone- and orexin-containing neurons. The results suggest that serotonin via postsynaptic 5-HT(1A) receptors affects the release of peptides regulating food intake.

Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake.

The neuropeptides orexin-A and orexin-B are produced in neurons of the lateral hypothalamic area and have been implicated to be involved in the regulation of food/water intake and sleep-wake control. The orexins act at two different G-protein-coupled orexin receptors (OX-R1 and OX-R2) that are derived from separate genes and expressed differentially throughout the central nervous system. In the present study, we have used a polyclonal antipeptide antiserum to analyse in detail the distribution of OX-R1-immunoreactive neurons in the rat hypothalamus. In order to identify the chemical mediators of orexin action in the hypothalamus, the OX-R1-containing neurons were characterized with regard to the content of peptides shown previously to affect ingestive and drinking behaviour. Neurons containing OX-R1 immunoreactivity were widely distributed in the hypothalamus with cell bodies located in the suprachiasmatic, periventricular, paraventricular (both magno- and parvocellular division), supraoptic, arcuate, ventromedial, dorsomedial and tuberomammillary nuclei and the lateral hypothalamic area. In magnocellular neurons of the paraventricular and supraoptic nuclei, OX-R1 immunoreactivity was seen in both vasopressin- and oxytocin-containing neurons. OX-R1 immunoreactivity was demonstrated in vasopressin and vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus, in somatostatin neurons of the periventricular nucleus and in corticotropin-releasing hormone (CRH) neurons of the parvocellular paraventricular nucleus. In the arcuate nucleus, OX-R1 immunoreactivity was present in neuropeptide Y (NPY) and agouti-related peptide (AGRP) neurons of the ventromedial part as well as in proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) neurons of the ventrolateral division. In the lateral hypothalamic area, OX-R1 immunoreactivity was demonstrated in melanin-concentrating hormone (MCH)- and orexin-containing neurons. In the hypothalamic tuberomammillary nucleus, OX-R1-immunoreactivity was shown in many histamine-containing neurons. The results support the idea that orexins have important actions on hypothalamic neurons that control food intake and fluid balance, but also that orexins may regulate other neuroendocrine systems.